CN117844758A - Method for separating and detecting circulating tumor cells - Google Patents
Method for separating and detecting circulating tumor cells Download PDFInfo
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Abstract
The application discloses methods for the isolation and detection of circulating tumor cells. Wherein, the method for separating the circulating tumor cells comprises the following steps: (i) Adding a combination of red blood cell specific antibodies and white blood cell specific antibodies to the blood sample to couple together red blood cells and white blood cells in the blood sample; (ii) Removing the red blood cells and white blood cells coupled together in the sample obtained in the step (i); (iii) Adding a red blood cell-specific antibody and a platelet-specific antibody to the sample obtained in step (ii); and (iv) further removing the red blood cell-specific antibodies, the platelet-specific antibodies, and the red blood cells and platelets bound thereto from the sample obtained in step (iii).
Description
Technical Field
The application relates to the technical field of biological macromolecule separation and detection, in particular to a single Circulating Tumor Cell (CTC) separation, purification and grabbing technology.
Background
CTCs are extremely rare populations of cells in the blood of cancer patients whose presence and number are closely related to the progression of tumor metastasis. However, since the number is very small and difficult to capture, previous studies have been limited to detection of surface markers, limiting our understanding of CTC molecular characteristics.
Single CTC cell RNA sequencing is achieved primarily through the following technical pathway: 1) Cell enrichment and identification and mechanical grasping: this is a critical step in the extraction of CTCs, usually by means of microfluidic techniques or immunoenrichment techniques. For example, a microfluidic may capture CTCs that meet specific physical or biological characteristics; antibodies are often used for immune enrichment, such as positive enrichment, a fragment avidin antibody (EpCAM) is used for capturing CTC of which the surface expresses a specific antigen, and then mechanical arm grabbing is performed through precise positioning under a microscope; 2) RNA sequencing technology: this involves sequencing RNA samples using high throughput sequencing. This process requires pretreatment of the RNA sample, such as reverse transcription into cDNA, followed by library creation and sequencing steps to generate sequencing data.
There are still some problems and disadvantages associated with the current state of the art. For example, existing CTC enrichment techniques, particularly those based on CTC biomarker positive enrichment, cannot obtain all CTCs because some CTCs may not express the selected marker and in some cases may misidentify healthy cells as CTCs. Although CTC negative enrichment can help to reduce interference of non-CTC cells to a certain extent, it still has some limitations and technical challenges, including residual red blood cells and platelets interfering with grasping and analysis of downstream CTC single cells, and at the same time, the experimental procedure of CTC identification and localization of the existing method causes the integrity of nucleic acid substances of CTCs to be destroyed, and how to compromise the activity of the cells to be tested to ensure the integrity of RNA has been a major challenge.
Therefore, there is a need for an efficient single cell grasping procedure for CTC purification enrichment and active CTCs to remedy the shortcomings of this technology.
Disclosure of Invention
To solve the above technical problem, an aspect of the present application provides a method for separating circulating tumor cells, the method comprising:
(i) Adding a combination of red blood cell specific antibodies and white blood cell specific antibodies to the blood sample to couple together red blood cells and white blood cells in the blood sample;
(ii) Removing the red blood cells and white blood cells coupled together in the sample obtained in the step (i);
(iii) Adding a red blood cell-specific antibody and a platelet-specific antibody to the sample obtained in step (ii); and
(iv) Further removing the red blood cell-specific antibodies, platelet-specific antibodies and red blood cells and platelets bound thereto from the sample obtained in step (iii).
In another aspect, the invention provides a method of detecting circulating tumor cells, the method comprising:
a. isolating circulating tumor cells by a method of isolating circulating tumor cells, the method of isolating comprising:
(i) Adding a combination of red blood cell specific antibodies and white blood cell specific antibodies to the blood sample to couple together red blood cells and white blood cells in the blood sample;
(ii) Removing the red blood cells and white blood cells coupled together in the sample obtained in the step (i);
(iii) Adding a red blood cell-specific antibody and a platelet-specific antibody to the sample obtained in step (ii); and
(iv) Further removing red blood cell specific antibodies, platelet specific antibodies and red blood cells and platelets bound thereto from the sample obtained in step (iii);
b. labeling the cells obtained in step a with a cell fluorescent antibody; and
c. detecting the fluorescence signal of the cells obtained in the step b.
Drawings
The present application is described in more detail below with reference to the attached drawing figures, wherein:
FIG. 1 is a schematic structural diagram of a method for detecting CTC negative enrichment according to the present invention;
FIG. 2 is a diagram showing an example of cell residue in the conventional method and the method of the present application;
FIG. 3 is a diagram showing an example of cell residue in the conventional method and the method of the present application;
FIG. 4 is a graph showing the comparison of the total number of cell residues in the conventional method and the method of the present application;
FIG. 5 is a graph showing the results of linear regression analysis of all sample result data in example 2;
FIG. 6 is a graph showing the detection rate results of all samples in example 2.
Detailed Description
The present application relates to a method of isolating circulating tumor cells comprising (i) adding a combination of red blood cell specific antibodies and white blood cell specific antibodies to a blood sample to couple red blood cells and white blood cells together in the blood sample. In one embodiment of the present application, the blood sample in step (i) comprises a fresh whole blood sample. In one embodiment of the present application, the blood sample in step (i) comprises a whole blood sample within 6 hours after blood collection. In this application, the erythrocyte-specific antibody may be any antibody that specifically binds to erythrocytes and promotes the coupling of erythrocytes to leukocytes after binding. In the present application, the leukocyte-specific antibody may be any antibody that specifically binds to leukocytes and promotes coupling of leukocytes with erythrocytes after binding. In this application, coupling may include any operation that brings red blood cells and white blood cells together to facilitate subsequent removal. In one embodiment of the present application, the combination of the erythrocyte-specific antibody and the leukocyte-specific antibody is provided in the form of a kit. In one embodiment of the present application, step (i) comprises incubating the blood sample after mixing with a combination of red blood cell specific antibodies and white blood cell specific antibodies. In one embodiment of the present application, the incubation temperature comprises 0-37 ℃. In one embodiment of the present application, the incubation temperature comprises room temperature. In one embodiment of the present application, the incubation time is from 1 minute to 1 hour. In one embodiment of the present application, the incubation time is about 5 minutes.
The method for isolating circulating tumor cells further comprises (ii) removing the coupled red blood cells and white blood cells from the sample obtained in step (i). In one embodiment of the present application, step (ii) comprises density gradient centrifugation. In one embodiment of the present application, step (ii) comprises adding the sample obtained in step (i) to a centrifuge tube with an insert, and obtaining a sample portion above the insert after centrifugation. In one embodiment of the present application, the insert includes a baffle having micro-holes. In one embodiment of the present application, the baffle comprises a plastic baffle. In one embodiment of the present application, the insert is transparent.
The method for isolating circulating tumor cells further comprises (iii) adding a red blood cell-specific antibody and a platelet-specific antibody to the sample obtained in step (ii). In one embodiment of the present application, the erythrocyte-specific antibody comprises an antibody against CD235 a. In one embodiment of the present application, the platelet-specific antibodies include antibodies against CD41a, CD42b and/or CD42 d. In one embodiment of the present application, the red blood cell-specific antibodies and platelet-specific antibodies in step (iii) comprise antibodies against CD41a, CD42b, CD42d and/or CD235 a. In a preferred embodiment of the present application, the red blood cell-specific antibodies and platelet-specific antibodies in step (iii) include antibodies against CD41a, CD42b, CD42d and CD235 a. In one embodiment of the present application, the red blood cell-specific antibodies and platelet-specific antibodies in step (iii) are labeled with biotin, and step (iv) comprises using microspheres comprising streptavidin on the surface. In one embodiment of the present application, the microspheres comprise magnetic beads. In one embodiment of the present application, step (iii) comprises incubating the sample after mixing with the erythrocyte-specific antibody and the platelet-specific antibody. In one embodiment of the present application, the incubation temperature comprises 0-37 ℃. In one embodiment of the present application, the incubation temperature comprises room temperature. In one embodiment of the present application, the incubation time is from 1 minute to 1 hour. In one embodiment of the present application, the incubation time is about 5 minutes. In a preferred embodiment of the present application, incubating comprises standing at room temperature for about 5 minutes.
The method for isolating circulating tumor cells further comprises (iv) further removing the red blood cell-specific antibodies, platelet-specific antibodies, and red blood cells and platelets bound thereto from the sample obtained in step (iii). In one embodiment of the present application, the microsphere comprises a magnetic bead and step (iv) comprises separating the magnetic bead using a magnet, e.g. a magnetic rack. In one embodiment of the present application, step (iv) comprises incubating the sample after mixing with the magnetic beads. In one embodiment of the present application, the incubation temperature comprises 0-37 ℃. In one embodiment of the present application, the incubation temperature comprises room temperature. In one embodiment of the present application, the incubation time is from 1 minute to 1 hour. In one embodiment of the present application, the incubation time is about 5 minutes. In a preferred embodiment of the present application, incubating comprises standing at room temperature for about 5 minutes.
The present application also relates to a method for detecting circulating tumor cells, comprising a. Isolating circulating tumor cells by an isolation method. In one embodiment of the present application, the separation method comprises the separation method of any one of the preceding embodiments.
The method for detecting the circulating tumor cells further comprises b, labeling the cells obtained in the step a with a cell fluorescent antibody. In one embodiment of the present application, the cytofluorescent antibodies include cell surface epithelial cell adhesion molecule antibodies against human EpCAM, cytokeratin antibodies against human CK, and/or leukocyte specific antibodies against human CD 45. In one embodiment of the present application, step b further comprises adding a nuclear dye. In one embodiment of the present application, the nuclear dye comprises DAPI. In a preferred embodiment of the present application, the cytofluorescent antibodies comprise cell surface epithelial cell adhesion molecule antibodies against human EpCAM, cytokeratin antibodies against human CK and leukocyte specific antibodies against human CD45, and step b further comprises the addition of the nuclear dye DAPI. In one embodiment of the present application, step b comprises incubating and staining. In one embodiment of the present application, step b comprises incubation in the absence of light. In one embodiment of the present application, the incubation temperature comprises 0-8 ℃. In one embodiment of the present application, the incubation temperature comprises about 4 ℃. In one embodiment of the present application, the incubation time is from 10 minutes to 2 hours. In one embodiment of the present application, the incubation time is about 30 minutes.
The method for detecting the circulating tumor cells further comprises c, detecting the fluorescent signals of the cells obtained in the step b. In one embodiment of the present application, circulating tumor cells are identified based on cell staining. In a preferred embodiment of the present application, cells exhibiting DAPI+ and CK/EpCAM+ and CD 45-staining characteristics are identified as circulating tumor cells. In one embodiment of the present application, step c further comprises centrifuging the cells obtained in step b. In one embodiment of the present application, step c comprises detection in a 96-well readout plate. In one embodiment of the present application, step c includes scanning and image analysis.
Some terms in this application are explained as follows:
1) CD41a: expressed on platelets and megakaryocytes for platelet activation and aggregation.
2) CD42a: expression is on platelets and megakaryocytes. Is involved in platelet adhesion and aggregation, and amplifies platelet response to thrombin.
3) CD42b: expressed on platelets and megakaryocytes. For platelet adhesion and aggregation.
4) CD42d: expression is on platelets and megakaryocytes. For platelet adhesion and aggregation.
5) CD235a: expression is on erythrocytes. The primary membrane-associated proteins of erythrocytes minimize erythrocyte aggregation.
6) CD45: leukocyte common antigen (Cluster of differentiation 45), all the leukocytes express, is a kind of transmembrane protein with larger molecular weight.
7) EpCAM: epithelial cell adhesion molecule (Epithelial cell adhesion molecule), cancer cell surface marker of epithelial origin.
8) CK is an abbreviation for cytokeratin, which is one of the CK family members and is widely distributed in cells. CK is commonly used to identify tumor cells of epithelial origin.
9) DAPI is an abbreviation for 4', 6-diamidino-2-phenylindole, a fluorescent dye capable of binding strongly to DNA, and is commonly used in fluorescent microscopy.
The following description of the embodiments of the present application will be made clearly and fully with reference to the accompanying drawings, in which some, but not all embodiments of the present application are shown. All other embodiments, based on the embodiments herein, which are within the scope of the protection of the present application, will be within the purview of one of ordinary skill in the art without the exercise of inventive faculty.
Examples
The technical scheme of the invention will be described in more detail below with reference to specific examples. The following examples are only examples, and do not constitute any limitation or restriction on the technical solution of the present invention. The specific materials, steps, conditions, values or numerical ranges specified in the following examples are illustrative only and are not intended to be exhaustive or limiting.
In addition to the specific methods, devices, materials used in the embodiments, any methods, devices, and materials of the prior art similar or equivalent to those described in the embodiments of the present invention may be used to practice the present invention according to the knowledge of one skilled in the art and the description of the present invention.
Table 1: list of reagents used
Table 2: list of instrument consumables for part
| Instrument for measuring and controlling the intensity of light | Manufacturer/goods number |
| CentrifugingMachine for making food | Eppendorf：5943000011 |
| Centrifuge tube | Corning：430791 |
| Vortex mixer | Hangzhou Oreg Cheng Yiqi Co., ltd: OS-100 |
| Fluorescent scanner | PerkinElmer Operetta CLS：HH16000020 |
| ALS CellCelector | ALS：AND-B723-01A |
| EDTA-2K anticoagulation tube | BD：367525 |
| Microscope | Olinbas: AX8922 and 8922 03 IX73 INST C20180402 |
In order to more clearly illustrate the technical scheme of the invention, the invention will be further described with reference to the embodiment.
Example 1: enrichment and isolation of CTC
A. Method for enriching and purifying single circulation tumor cells
Step one: CTC enrichment
Referring to FIG. 1, a schematic structural diagram of the CTC negative enrichment detection method of the present invention is shown. As shown in fig. 1, the method for detecting the negative enrichment of the circulating tumor cells comprises the following steps:
first, step S1 is performed by adding a solution a (sample density separating solution, 16 persons/box, courser biotechnology (Shanghai) limited) to a first centrifuge tube containing a whole blood sample, and coupling red blood cells and white blood cells in the whole blood sample. And then removing the red blood cells and the white blood cells which are coupled together in the whole blood sample by using a density gradient centrifugation method, so that the negative enrichment of the CTC cells is realized. The method specifically comprises the following steps:
placing the first centrifuge tube on a shaking table for incubation;
adding liquid B (sample density separating liquid, 16 persons/box, jun-real biotechnology (Shanghai) limited company) into a second centrifuge tube with an insert, and transferring the incubated whole blood sample in the first centrifuge tube into the second centrifuge tube;
placing the second centrifuge tube in a centrifuge for centrifugation, wherein a monocyte layer in the whole blood sample is positioned above the plug-in unit after centrifugation, and red blood cells and white blood cells which are coupled together are positioned below the plug-in unit;
and pouring the sample which is subjected to centrifugation and is positioned above the insert in the second centrifuge tube into a third centrifuge tube, namely pouring the mononuclear cell layer which is positioned above the insert into the third centrifuge tube.
The plug-in unit is a transparent plastic baffle with micropores in the middle, red blood cells are arranged below the plug-in unit after density gradient centrifugation, a monocyte layer is arranged above the plug-in unit, the density gradient centrifuge tube can be directly inverted, liquid above the plug-in unit is completely poured out, and a red blood cell layer sample below the plug-in unit is blocked; compared with a gun head for sucking the monocyte layer sample, the centrifuge tube with the plug-in is convenient for a user to extract the monocyte sample, and can maximally ensure the monocyte collecting efficiency.
Step two: single CTC cell purification
In order to implement the red blood cell, platelet removal and cell fluorescence staining method of the present invention, step S2 is performed, operating as follows:
after centrifugation of the tube, the supernatant was discarded using a 51L pipette, leaving about 1mL of the bottom residual liquid. This step should be performed slowly so as not to disturb the cells at the bottom.
To each centrifuge tube was added 4. Mu.l each of the CD41a, CD42a, CD42b, CD42d, CD235a biotin-labeled antibodies, and sealed (blown in a blocking solution) with a blocking tip. Then gently blowing and mixing for 3-5 times, and standing for 5 minutes at room temperature. After the incubation was completed, a cell dilution at 4℃was added to dilute the sample to 2 times the original volume.
Streptavidin T1 magnetic beads (Dynabeads MyOne) were vortexed on a vortex mixer for 30 seconds, then the same amount of magnetic beads as the removal solution was aspirated, and added to the above samples. After being blown and evenly mixed for 3-5 times by using a pipetting gun, the samples are placed on a magnetic rack and incubated for 5 minutes at room temperature.
After incubation was completed, the supernatant (note not to be attracted to magnetic beads) was transferred to a new 14mL round bottom centrifuge tube using a pipette.
All samples were again placed on an adaptive magnet rack and incubated for 5 minutes at room temperature to allow for magnetic bead adsorption. Subsequently, the supernatant (note not to attract the beads) was transferred to a new 14mL conical centrifuge tube.
PBS wash was added to 14.5mL to centrifuge the sample, leaving about 100 μl of the centrifuge supernatant.
B. Method for staining circulating tumor cells
Next, step S3 is performed to label CTCs by suspension staining with a cell fluorescent antibody, and CTCs are identified by cell staining and morphology based on the fluorescent signal collected by scanning. The added reagents include: cell surface epithelial cell adhesion molecule antibodies against human EpCAM, cytokeratin antibodies against human CK, nuclear dye DAPI, and leukocyte specific antibodies against human CD45 (circulating tumor cell staining fluid, 16 parts per box, courser biotechnology (shanghai) inc.).
In the implementation S3, we collected the fluorescent signal of the cells by a fluorescent scanner and identified CTCs based on staining and morphological features of the cells. In this example, we define cells meeting the following characteristics as CTCs: nuclei display DAPI fluorescence (dapi+) and CK or EpCAM display fluorescence (CK/epcam+), whereas CD45 does not display fluorescence (CD 45 "). Here, the symbol "+" indicates that there is a fluorescent signal, i.e., CK/epcam+ indicates that CK or EpCAM exhibits a fluorescent signal; and the symbol "-" indicates no fluorescent signal.
Further, the specific steps of executing S3 are as follows:
1. adding cell diluent into the third centrifugal tube, sequentially and uniformly mixing, and then performing 800x g centrifugation;
2. discarding the supernatant, adding the nuclear dye, cytokeratin, epCAM and CD45 into a third centrifuge tube, and incubating and staining in a dark place; this operation was performed in a refrigerator at 4℃and stained incubation was performed for 0.5 hours, during which the mixture was mixed by shaking manually every 10 minutes.
3. Replenishing cell washing liquid again, and centrifuging;
4. the supernatant was discarded and the sample in the third centrifuge tube was transferred to a 96 well reading plate for scanning and image analysis.
5. The sample after scanning is immediately grabbed target CTC single cells through an ALS cell selector to carry out single cell RNA sequencing, and the sample can be placed in a refrigerator at-80 ℃ for a long time for preservation.
In view of the above, the method for detecting the negative enrichment of the circulating tumor cells of the present application employs that a combination of a red blood cell specific antibody and a white blood cell specific antibody is first added to a whole blood sample to adsorb red blood cells and white blood cells, and most of the red blood cells and white blood cells in the blood sample are removed by a density gradient centrifugation method, and then the specific antibody combined with platelets and red blood cells and corresponding adsorption magnetic beads are added. Effectively solves the problem that platelets and erythrocytes cannot be removed completely, thereby influencing the high-purity extraction of downstream CTC single cells. On the other hand, the invention adopts a solution that all consumables (such as test tubes, gun heads, 96-well plates and the like) contacted with the whole blood sample are subjected to anti-cell adsorption treatment before use, so that the risk of cell loss in the operation process is reduced, and the sensitivity of a circulating tumor cell detection and sequencing method based on negative enrichment is further improved.
EXAMPLE 2 demonstration of erythrocyte and platelet removal Effect and cell viability
For a more thorough understanding of the methods of the present application, the following examples provide detailed details for the detection of negative cells enriched from circulating tumor cells:
we take EpCAM/CK-expressing tumor cells (e.g., human breast cancer SKBR3 cells) as an example, these cells were incorporated into a healthy human blood sample collected using EDTA-2K anticoagulant tubes for analytical performance evaluation. The number of incorporated SKBR3 cells was accurately counted by microscopy.
The method is suitable for clinical whole blood samples collected by EDTA-2K anticoagulant tubes.
The same blood sample was divided into two groups. A set of platelet and erythrocyte removal methods using the present application, comprising the following specific steps:
first, a first centrifuge tube (approximately 5mL volume containing whole blood and liquid a (sample density isolate, 16 persons/cassette, courser biotechnology (Shanghai) limited)) was placed on a shaker for incubation.
Liquid B (sample density separation liquid, 16 persons/box, courser biotechnology (shanghai) limited) was added to a second centrifuge tube with an insert, and the whole blood sample in the incubated first centrifuge tube was transferred to the second centrifuge tube.
The second centrifuge tube is placed in a centrifuge for centrifugation, after which the mononuclear cell layer in the whole blood sample is above the insert and the coupled red blood cells and white blood cells are below the insert.
And pouring the sample which is positioned above the insert in the second centrifuge tube after centrifugation into a third centrifuge tube, namely pouring the mononuclear cell layer positioned above the insert into the third centrifuge tube.
The insert is a transparent plastic baffle with micro-holes in the middle, and after density gradient centrifugation, the red blood cells will be located under the insert, while the monocyte layer will be located over the insert. The density gradient centrifuge tube can be directly inverted, the liquid above the plug-in unit is completely poured out, and the erythrocyte layer sample below the plug-in unit is blocked. Compared with the method that a gun head is used for sucking the mononuclear cell layer sample, the centrifuge tube with the plug-in is convenient for a user to extract the mononuclear cell sample, and the mononuclear cell collecting efficiency can be guaranteed to the greatest extent.
After centrifugation of the tube, the supernatant was discarded using a 5mL pipette, leaving about 1mL of the bottom residual liquid. This step should be performed slowly so as not to disturb the cells at the bottom.
To each centrifuge tube was added 4. Mu.l each of the CD41a, CD42a, CD42b, CD42d, CD235a biotin-labeled antibodies, and sealed (blown in a blocking solution) with a blocking tip. Then gently blowing and mixing for 3-5 times, and standing for 5 minutes at room temperature. After the incubation was completed, a cell dilution at 4℃was added to dilute the sample to 2 times the original volume.
Streptavidin T1 magnetic beads (Dynabeads MyOne) were vortexed on a vortex mixer for 30 seconds, then the same amount of magnetic beads as the removal solution was aspirated, and added to the above samples. After being blown and evenly mixed for 3-5 times by using a pipetting gun, the samples are placed on a magnetic rack and incubated for 5 minutes at room temperature.
After incubation was completed, the supernatant (note not to be attracted to magnetic beads) was transferred to a new 14mL round bottom centrifuge tube using a pipette.
All samples were again placed on an adaptive magnet rack and incubated for 5 minutes at room temperature to allow for magnetic bead adsorption. Subsequently, the supernatant (note not to attract the beads) was transferred to a new 14mL conical centrifuge tube.
PBS wash was added to 14.5mL to centrifuge the sample, leaving about 100 μl of the centrifuge supernatant.
Adding nuclear dye, cytokeratin, epCAM and CD45 (circulating tumor cell staining solution, 16 parts/box, courser biotechnology (Shanghai) limited) to a third centrifuge tube, incubating and staining in the absence of light; this operation was performed in a refrigerator at 4℃and stained incubation was performed for 0.5 hours, during which the mixture was mixed by shaking manually every 10 minutes.
Replenishing cell washing liquid again, and centrifuging;
the supernatant was discarded and the sample in the third centrifuge tube was transferred to a 96 well reading plate for scanning and image analysis.
The sample after scanning is immediately grabbed target CTC single cells through an ALS cell selector to carry out single cell RNA sequencing, and the sample can be placed in a refrigerator at-80 ℃ for a long time for preservation.
Another group uses the conventional method, and the specific operation flow is as follows:
first, a first centrifuge tube (approximately 5mL volume containing whole blood and liquid a (sample density isolate, 16 persons/cassette, courser biotechnology (Shanghai) limited)) was placed on a shaker for incubation.
Liquid B (sample density separation liquid, 16 persons/box, courser biotechnology (shanghai) limited) was added to a second centrifuge tube with an insert, and the whole blood sample in the incubated first centrifuge tube was transferred to the second centrifuge tube.
The second centrifuge tube is placed in a centrifuge for centrifugation, after which the mononuclear cell layer in the whole blood sample is above the insert and the coupled red blood cells and white blood cells are below the insert.
And pouring the sample which is positioned above the insert in the second centrifuge tube after centrifugation into a third centrifuge tube, namely pouring the mononuclear cell layer positioned above the insert into the third centrifuge tube.
The insert is a transparent plastic baffle with micro-holes in the middle, and after density gradient centrifugation, the red blood cells will be located under the insert, while the monocyte layer will be located over the insert. The density gradient centrifuge tube can be directly inverted, the liquid above the plug-in unit is completely poured out, and the erythrocyte layer sample below the plug-in unit is blocked. Compared with the method that a gun head is used for sucking the mononuclear cell layer sample, the centrifuge tube with the plug-in is convenient for a user to extract the mononuclear cell sample, and the mononuclear cell collecting efficiency can be guaranteed to the greatest extent.
Cell dilutions were added to a third centrifuge tube containing supernatant to 14.5mL, then slowly turned upside down 8 times, and the solution was mixed. Centrifuge at 1500rpm for 10min.
The third tube was centrifuged to discard the supernatant, leaving about 100. Mu.L of liquid at the bottom, and the action was slow and the bottom cells were not disturbed.
200. Mu.L of cell fixative (Centipede, FD 03273) was added to the tube, the tips were closed, and the mixture was blown and mixed, allowed to stand at room temperature for 15min, then 5mL of cell wash was added, and the mixture was placed in a centrifuge for centrifugation at 1200rpm for 10min.
The supernatant was discarded to 100. Mu.L, and nuclear dye, cytokeratin, epCAM and CD45 (circulating tumor cell staining solution, 16 parts/box, courser Biotechnology (Shanghai) Co.) were added to the third centrifuge tube, incubated and stained at room temperature in the absence of light, and stained for 1 hour.
Replenishing cell washing liquid again, and centrifuging;
the supernatant was discarded, and the samples in the third centrifuge tube were transferred to a 96-well reading plate, settled for 1 hour, and then scanned and image analyzed.
The conventional method has the defects that the residual red blood cells and platelets have huge densities, so that the cells are stacked together and single-cell grabbing cannot be performed.
We will compare the total number of blood cells treated by the two methods with the total cell viability.
1. Verification scheme
Over three consecutive days we treated each day with the conventional and inventive methods, respectively, 4 blood samples from the donor. Each blood sample was divided equally into aliquots, one of which was treated by the method of the invention and the other by conventional methods. The total number of cells was compared between the conventional method and the method of the present invention, and the total cell viability was recorded to draw conclusions.
Cell total activity measurement method: mu.L of the cell sample was mixed with 5. Mu.L of ReadyCount (Invitrogen ReadyCount "Green/Red Cell Viability Stain, A49005) stain and after incubation for 5 minutes at room temperature, 10. Mu.L of the stained cell sample was transferred to a 96 well readout plate for scanning counting and image analysis under FITC and TRITC channels.
Cell viability calculation formula = cell number of FITC +/(total cell amount of FITC + and TRITC +)%
2. Result determination
If the total number of residual cells in the final method is less than 3000 and the average value of the total cell activity is more than 80 percent, the requirement of a verification scheme is met.
3. Test results
For comparison of the number of platelets in the samples of the conventional and inventive methods, refer to FIGS. 2, 3 and Table 4. FIG. 2 illustrates a sample treated by the conventional method and the method of the present invention, in the case of bright field, with platelets removed. FIG. 3 randomly illustrates the removal of platelets from samples treated by conventional methods and methods of the present invention, in the case of specific staining of platelet surface antigens with FITC-labeled CD41 antibodies (green fluorescent staining shown). Table 4 statistics and calculations are made on the number of platelets in the sample exemplified in fig. 3, as well as the removal rate. For the total number of cell residues in the conventional method and the method of the present invention, refer to the comparison result of FIG. 4. For the results of total cell viability, please refer to table 3. By comparison, we can draw conclusions about the platelet, erythrocyte removal effect of the method of the invention, and the overall cell viability results associated.
Table 3: total cell viability after removal of erythrocytes and platelets by the methods and conventional methods of the invention
Table 4: the method of the invention removes the platelet efficiency result
| Sample number | Removal of Pre-platelet count | Platelet count after removal | Removal rate of |
| 1 | 5384 | 57 | 99％ |
| 2 | 6252 | 110 | 98％ |
| 3 | 6155 | 69 | 99％ |
Verifying accuracy and determining measurement range
We evaluate the accuracy of the detection method by recovery experiments. Specifically, we added a known number of SKBR3 cells to the negative stromal blood to assess whether the assay method was able to accurately determine the number of cells added. We expressed the results in terms of recovery, which was obtained by measuring the ratio of the number of positive cells recovered to the number of actual incorporated cells after varying numbers of incorporated cells (from 0 to 100), and multiplying by 100%. By calculating the recovery of the different numbers of incorporated cells we have obtained the average recovery and by performing a linear regression analysis we have determined the optimal measurement range of the method.
1. Verification scheme
Over three consecutive days we added approximately 100, 50, 10 and 0 SKBR3 cells each day to 5 blood samples (5 ml) of healthy donors. Subsequently, we observed the recorded number of tumor cells, divided by the corresponding number of tumor cells added, resulting in recovery. The average of these recoveries was taken as the overall average recovery. By performing linear regression analysis, we determined the measurement range of the CTC negative enrichment detection method.
2. Result determination
If the average recovery exceeds 60%, the requirements of the verification scheme are satisfied. Where the desired recovery can be achieved within a linear range, the measurement range of the method can be determined.
3. Test results
For average recovery data for samples of each incorporated cell group, please refer to table 5; fig. 5 shows the results of linear regression analysis of all sample result data.
Table 5: average recovery data for all samples each incorporated into the following group
A total of three validation experiments were performed and the results of the experiments are recorded in table 5. In these test results, the lowest sample recovery was 64%, while the average recovery for all samples was higher than 66%.
Subsequently, the result data of all samples were subjected to linear analysis, and the specific results are shown in fig. 5. The linear regression equation is: y= 0.7796x-1.1059, and the correlation coefficient R2 is 0.97. In the range of 0-100 cells, the recovery rate data of all samples exceeds 60%, and the linear regression line fitting goodness is good. Thus, the method is currently applicable to a measurement range of 0-100 cells.
Minimum detection limit verification
Adding different numbers of SKBR3 cells into negative matrix blood samples for detection, calculating the detection rate of each sample, and selecting the lowest cell level with the number of detected circulating tumor cells being more than or equal to 5 as the lowest detection limit.
1. Verification method
Approximately 10, 8 and 5 SKBR3 cells were added to the stromal blood samples (5 ml), respectively, and repeated 10 times in each case. The minimum cell level at which the recovery rate data of the sample exceeds 60% is finally used as the minimum detection limit.
2. Result determination
The detection rate of the number of each added cell was calculated separately, and the lowest cell level at which the positive detection rate reached 100% was determined as the lowest detection limit.
3. Experimental results
The lower limit of detection experiments were performed over 3 time periods, with a total of 3 experiments performed with about 10, 8, and 5 SKBR3 cells added at a time, and the results are shown in fig. 6. When more than 8 cells are added, cell recovery can reach at least 60%.
In summary, compared with the prior art, the invention has the following advantages:
1. based on the circulating tumor cell negative enrichment detection method, the purity and the survival rate of CTC cells in a final sample can be effectively improved.
2. The method effectively reduces the complexity of downstream experimental work such as subsequent identification, single-cell RNA sequencing and the like, and improves the efficiency and accuracy of identification and single-cell RNA sequencing.
The foregoing is merely a specific application example of the present application, and the protection scope of the present application is not limited in any way. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary or obvious to describe all embodiments. All such technical solutions formed by equivalent transformation or equivalent substitution fall within the protection scope of the claims of the present application.
Claims (10)
1. A method of isolating circulating tumor cells, the method comprising:
(i) Adding a combination of red blood cell specific antibodies and white blood cell specific antibodies to the blood sample to couple together red blood cells and white blood cells in the blood sample;
(ii) Removing the red blood cells and white blood cells coupled together in the sample obtained in the step (i);
(iii) Adding a red blood cell-specific antibody and a platelet-specific antibody to the sample obtained in step (ii); and
(iv) Further removing the red blood cell-specific antibodies, platelet-specific antibodies and red blood cells and platelets bound thereto from the sample obtained in step (iii).
2. The separation method of claim 1, wherein the blood sample in step (i) comprises a fresh whole blood sample, e.g., a whole blood sample within 6 hours after blood collection.
3. The separation method of claim 1, wherein step (ii) comprises density gradient centrifugation.
4. A separation method according to claim 3 wherein step (ii) comprises adding the sample from step (i) to a centrifuge tube with an insert and obtaining a portion of the sample above the insert after centrifugation.
5. The separation method of claim 4, wherein the insert comprises a baffle, preferably a plastic baffle, having micro-holes.
6. The separation method of claim 1, wherein the red blood cell-specific antibodies and platelet-specific antibodies in step (iii) comprise antibodies to CD41a, CD42b, CD42d and/or CD235 a.
7. The separation method of claim 1, wherein the red blood cell-specific antibodies and the platelet-specific antibodies in step (iii) are labeled with biotin, and step (iv) comprises using microspheres, e.g., magnetic beads, that contain streptavidin on their surface.
8. A method of detecting circulating tumor cells, the method comprising:
a. isolating circulating tumor cells by the isolation method of any one of claims 1-7;
b. labeling the cells obtained in step a with a cell fluorescent antibody; and
c. detecting the fluorescence signal of the cells obtained in the step b.
9. The assay of claim 8, wherein the cell fluorescent antibody comprises a cell surface epithelial cell adhesion molecule antibody against human EpCAM, a cytokeratin antibody against human CK and/or a leukocyte specific antibody against human CD45, and step b further comprises the addition of a nuclear dye, preferably DAPI.
10. The detection method according to claim 8, wherein the circulating tumor cells are identified on the basis of cell staining, preferably cells exhibiting dapi+ and CK/epcam+ and CD 45-staining characteristics are determined as circulating tumor cells.
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